Oxidative stress responsive apoptosis inducing protein eif5a

ABSTRACT

Secreted eIF5A protein which is useful for diagnosis, prevention, and treatment of various diseases induced by an oxidative stress.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 12/994,824 filed Feb. 23, 2011, which is incorporated herein byreference, which was a National Stage application of PCT/JP2009/02344,filed on May 27, 2009, and claims the benefit of Japanese patentapplication JP 2008-137824, filed on May 27, 2008.

TECHNICAL FIELD

The present invention relates to a protein which inhibits apoptosis, inparticular, apoptosis caused by oxidative stress, and a diagnostic agentand a pharmaceutical which contain the same.

BACKGROUND ART

It is known that mammalian cells quickly respond and adapt toenvironmental stimuli (for example, mechanical load, metabolic changes,ischemia and reperfusion) by expressing a number of genes. Especially,oxidative stress induced by various external stresses (for example,ischemia followed by reperfusion, ultraviolet burn, and irradiation) isknown to play a key role in pathogenesis of cell injury involved, whichaccelerates inflammation, atherosclerosis, aging, and the like.

In particular, cardiac myocytes express various genes coding for growthfactors, cytokines, cell-adhesion molecules, and so on, in response toischemia/reperfusion to adapt to these stresses, or lead to further celldamage known as reperfusion injury. The threshold of cardiac myocytes toundergo apoptosis seems to be so high to protect these non-divisioncells from external stresses. For example, only small part of cardiacmyocytes can undergo apoptosis even in such a situation of acutemyocarditis in which strong expression of Fas on cardiac myocytes andFasL on infiltrating lymphocytes was induced (Non Patent Literature 1).However, there is the only exception of the case, which is reperfusioninjury. Reperfusion of ischemic tissue causes massive production ofoxygen free radicals, excessive intracellular calcium influx, andneutrophil infiltration, resulting in acute inflammation associated withextensive apoptosis of cells. Because reperfusion-induced apoptotic celldeath was not induced by ischemia alone, and could not be prevented byneutrophil depletion (Non Patent Literature 2), it has been proposedthat some mechanism triggered by reperfusion mediates the apoptosissignaling pathway, which may precede and be independent of neutrophilinfiltration (Non Patent Literature 3).

Eukaryotic translation initiation factor (eIF) 5A is a substance whichwas identified as a translation initiation factor as its name suggests.It is known that eIF5A is expressed in the cytoplasm, deoxyhypusinatedby deoxyhypusine synthase (DHS) (deoxyhypusinated eIF5A), and after thathypusinated by deoxyhypusine hydroxylase (DOHH) (hypusinated eIF5A), andthat this hypusinated eIF5A exhibits a cell proliferative action (NonPatent Literature 4). However, it has not been known at all that eIF5Ais secreted extracellularly, and what role the secreted eIF5A plays ininduction of apoptosis.

CITATION LIST Non Patent Literature

-   [Non Patent Literature 1] J. Am. Coll. Cardiol. 39, 1399-1403 (2002)-   [Non Patent Literature 2] J. Clin. Invest. 94, 1621-1628 (1994)-   [Non Patent Literature 3] Am. J. Pathol. 151, 1257-1263 (1997)-   [Non Patent Literature 4] Amino Acids 20, 91-104 (2001)

SUMMARY OF INVENTION Problem to be Solved by the Invention

An object of the present invention is to provide an agent which isuseful for diagnosis, prevention, and treatment of various diseasesinduced by oxidative stress.

Solution to Problem

Accordingly, the inventors of the present invention adopted culturedcells which were subjected to hypoxia/reoxygenation as an in vitro modelof ischemia followed by reperfusion, in which cell injury, that is,apoptosis is known to be caused by oxidative stress. The inventors ofthe present invention considered that a certain humoral factor releasedfrom cultured cells exposed to the hypoxia/reoxygenation condition isinvolved in apoptosis and analyzed the humoral factor. As a result, theinventors of the present invention found that eIF5A, which has beenheretofore reported to be present only in the cytoplasm, is secretedextracellularly from cells exposed to the hypoxia/reoxygenationcondition, that is, under an oxidative stress condition, and that thesecreted eIF5A is a novel protein as it clearly differs from the eIF5Apresent in the cytoplasm with regards to the isoelectric point.

Furthermore, the inventors of the present invention found that when acell is exposed to hypoxia/reoxygenation, that is, an oxidative stress,the secreted eIF5A is secreted extracellularly, acts as anapoptosis-inducing ligand, and induces apoptosis of the cells exposed tooxidative stress, and hence oxidative stress can be diagnosed bymeasuring the secreted eIF5A.

Furthermore, the inventors of the present invention found that theapoptosis can be significantly inhibited by allowing an anti-eIF5Aneutralizing antibody to act as a representative substance whichinhibits secreted eIF5A. The present invention was thus accomplished.

Specifically, the present invention provides a secreted eIF5A protein.

The present invention further provides a pharmaceutical containing asecreted eIF5A protein.

The present invention further provides a method for determining anoxidative stress, including measuring a secreted eIF5A protein in afluid or a tissue specimen

The present invention further provides a diagnostic agent for oxidativestress, containing a reagent for measuring a secreted eIF5A protein.

The present invention further provides an apoptosis inhibitor,containing a secreted eIF5A protein inhibitor.

The present invention further provides use of a secreted eIF5A proteinfor production of an apoptosis inducer.

The present invention further provides use of a secreted eIF5A proteininhibitor for production of an apoptosis inhibitor.

The present invention further provides a method for inducing apoptosis,including administering a secreted eIF5A protein.

The present invention further provides a method for inhibitingapoptosis, including administering a secreted eIF5A protein inhibitor.

Advantageous Effects of Invention

As a secreted eIF5A protein of the present invention is an entirely newprotein which is secreted from a mammalian cell when the cell is exposedto an oxidative stress, an oxidative stress condition of mammalsincluding humans can be determined and diagnosed by measuring thesecreted eIF5A protein. Specifically, whether or not cell injury causedby an oxidative stress may occur or cell injury has already occurred canbe diagnosed.

Furthermore, as the secreted eIF5A protein induces not only apoptosiscaused by an oxidative stress, but apoptosis at a normoxicconcentration, the secreted eIF5A protein is useful as a therapeuticagent for cancer and diseases represented by infiltrative diseases.

Furthermore, as a secreted eIF5A protein inhibitor inhibits apoptosiscaused by an oxidative stress, the secreted eIF5A protein inhibitor isuseful as a preventive or therapeutic pharmaceutical for not onlyischemia/reperfusion injury and ultraviolet ray/radiation damage, butalso atherosclerosis, aging, and the like accelerated by an oxidativestress.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows chromatofocusing of reoxygenation-conditioned PBS (RCP;upper panel) and control-conditioned PBS (CCP; lower panel) fromcultured cardiac myocytes. Black bars indicate the ERK activatingactivity of each fraction on cultured cardiac myocytes.

FIG. 2 a shows two-dimensional gel electrophoresis of active fractions(fractions 49 to 52) from RCP (left panel) and CCP (right panel) stainedwith silver. Arrows in RCP (left panel) indicate protein spots (1 and2). Of these, a spot corresponding to the protein spot 1 is not presentin CCP (right panel). Only a small spot (2′) corresponding to 2 ispresent in CCP (right panel).

FIG. 2 b shows Western analysis of a cytosolic fraction of recombinanteIF5A from untreated transfected cells (cytosolic; left panel) andrecombinant eIF5A from RCP (right panel), blotted with anti-FLAG mAb.

FIG. 3 a shows induction of apoptosis of cultured cardiac myocytes dueto a recombinant eIF5A protein (10 μg/mL) as determined by doubleimmunostaining for TUNEL (brown color) and cardiac myosin (blue color).

FIG. 3 b shows time course of the percentages of apoptotic cardiacmyocytes determined by TUNEL staining induced by recombinant eIF5A(RCP), recombinant mutant eIF5A (K50A), and recombinant (cytosolic)eIF5A.

FIG. 3 c is a representative confocal image of the effect of recombinanteIF5A (RCP) on subcellular translocation of an apoptosis-inducing factor(AIF) from the cytoplasm to the nucleus.

FIG. 3 d shows Western blot analysis anti-cytochrome c mAb (7H8.2C12;Lab Vision Corp.) of the effects of recombinant eIF5A (RCP) oncytochrome c released from mitochondria.

FIG. 3 e shows Western blot analysis of the effect of recombinant eIF5A(RCP) on the activation of caspase-3 with anti-caspase-3 polyclonalantibody (H-277; Santa Cruz Biotechnology).

FIG. 3 f shows induction of apoptosis of cardiac myocytes as determinedby double immunostaining for annexin-V (upper panel, labeled with FITC)and cardiac myosin (lower panel, labeled with TRITC).

FIG. 3 g shows electron microscopic determination of hypercondensationof nuclear chromatin induced by recombinant eIF5A (RCP).

FIG. 3 h shows the effects of a PARP-1 inhibitor (3-aminobenzamide, 2mM) or caspase inhibitor (Z-VAD.fmk, 100 μM), added at 1 hour beforeaddition of recombinant eIF5A (RCP), on the induction of apoptosis ofcardiac myocytes by double immunostaining for TUNEL (brown color) andcardiac myosin (blue color) (P<0.001*: n=4).

FIG. 3 i shows the percentages of apoptotic cardiac myocytes, determinedby TUNEL staining at 72 hours after addition of recombinant eIF5A (RCP).Data are expressed as mean±S.D.

FIG. 3 j shows the effect of immunodepletion of eIF5A onreoxygenation-conditioned media (RCM)-induced apoptosis of cardiacmyocytes by double immunostaining for TUNEL (brown color) and cardiacmyosin (blue color) at 30 hours after adding RCM.

FIG. 3 k shows the effect of immunodepletion of eIF5A onreoxygenation-conditioned media (RCM)-induced apoptosis of cardiacmyocyte, as percentages of apoptotic cardiac myocytes, which weredetermined by double immunostaining for TUNEL and cardiac myosin at 30hours after addition of RCM.

FIG. 4 is a diagram showing two-dimensional electrophoresis results ofmyc-tagged and FLAG-tagged recombinant eIF5A. The upper panel showscytosolic eIF5A, and the lower panel shows secreted eIF5A. A isunhypusinated eIF5A, and B is hypusinated eIF5A.

FIG. 5 a shows immunofluorescence localization of an eIF5A protein incultured cardiac myocyte in response to hypoxia/reoxygenation determinedby double immunostaining with an anti-eIF5A antibody (J-M) and ananti-cardiac myosin (CMA19).

FIG. 5 b shows immunoelectron microscopic subcellular localization of aneIF5A protein in cultured cardiac myocyte in response tohypoxia/reoxygenation with an anti-eIF5A antibody (J-M) and ProteinA-labeled colloidal gold.

FIG. 5 c shows immunofluorescence of an eIF5A protein in myocardialtissues from sham-operated rats and rats subjected to myocardialischemia/reperfusion in vivo.

FIG. 6 is a diagram showing the apoptosis inducing ability (TUNELstaining) of recombinant eIF5A (re-eIF5A) on various cells.

FIG. 7 is a diagram showing changes with time in the apoptosis inducingability of recombinant eIF5A (re-eIF5A) on Hela cells.

FIG. 8 is a diagram showing the inhibitory effect of a DHS inhibitor(GC7) on rat myocardial ischemia perfusion injury. The proportion of theinfracted area to the whole cross section area of the myocardial tissueis shown on the right side.

FIG. 9 is a diagram showing the inhibitory effect of an anti-eIF5Aneutralizing monoclonal antibody on rat myocardial ischemia/reperfusioninjury. The proportion of the infracted area to the whole cross sectionarea of the myocardial tissue is shown on the right side.

FIG. 10 is a diagram showing serum level of secreted eIF5A in myocardialischemic reperfusion injury. The p value was calculated by paired t-test(n=5).

MODES FOR CARRYING OUT THE INVENTION

The secreted eIF5A protein of the present invention (hereinafter alsoreferred to as secreted eIF5A) is a novel protein which is secretedextracellularly from a cell exposed to an oxidative stress. ConventionaleIF5A is a protein which is said to be produced in the cytoplasm andchanged to deoxyhypusinated eIF5A by deoxyhypusine synthase and furtherto hypusinated eIF5A, an active form, by the action of deoxyhypusinehydroxylase. Furthermore, the action of the hypusinated eIF5A is said topromote translation of mRNA involved in cell proliferation (Amino Acids2001; 20: 91-104). However, the eIF5A is a protein which is present onlyin the cytoplasm and was not known to be secreted extracellularly.

The secreted eIF5A of the present invention clearly differs from theeIF5A which is present in the cytoplasm not only in function but also asa substance. Specifically, the isoelectric point of the cytoplasmiceIF5A is 5.5 to 5.4, whereas the isoelectric point of the secreted eIF5Ais 5.4 to 5.3. That is, the isoelectric point of the secreted eIF5A is0.1 lower than the isoelectric point of cytosolic eIF5A.

Furthermore, the secreted eIF5A of the present invention includes anunhypusinated form and a hypusinated form. The isoelectric point of thesecreted hypusinated eIF5A is approx. 5.4, which is approx. 0.1 higherthan that of the secreted unhypusinated eIF5A. The isoelectric pointmentioned herein is a value determined by two-dimensional gelelectrophoresis.

More specifically, among the secreted eIF5A proteins of the presentinvention, the isoelectric point of the secreted unhypusinated eIF5A isapprox. 5.3, which is approx. 0.1 lower than that of the cytosolicunhypusinated eIF5A. The isoelectric point of the secreted hypusinatedeIF5A is approx. 5.4, which is 0.1 higher than that of the secretedunhypusinated eIF5A.

Between the secreted unhypusinated eIF5A and the secreted hypusinatedeIF5A, the secreted hypusinated eIF5A has a higher apoptosis-inducingactivity and is particularly preferred.

As the secreted (hypusinated) eIF5A of the present invention issecreted, for example, from cultured cells exposed to ahypoxia/reoxygenation condition, the secreted (hypusinated) eIF5A can becollected from a medium of the cultured cells. The cultured cells usedherein are not particularly limited so long as the cells are culturablecells derived from mammals, and examples thereof include cardiacmyocytes as well as various established cell lines. Examples of thehypoxia/reoxygenation condition include culturing at an oxygenconcentration of lower than 0.1% for 20 to 60 min and after that at anormal oxygen concentration of 20%. After 10 to 15 min of culturingunder a reoxygenation condition, the secreted (hypusinated) eIF5A of thepresent invention can be separated from the culture supernatant.

Furthermore, as the cytosolic (hypusinated) eIF5A has already beencloned, the secreted (hypusinated) eIF5A of the present invention canalso be produced by culturing, under a hypoxia/reoxygenation condition,cells transfected with a eIF5A gene by recombinant DNA technology andcollecting the eIF5A from the culture supernatant. Here, the nucleotidesequence of the cytosolic eIF5A gene and the amino acid sequence of thecytosolic eIF5A are shown as SEQ ID NO: 1. As the secreted eIF5A of thepresent invention is also obtained by expressing the gene of the eIF5A,the secreted eIF5A has the amino acid sequence of SEQ ID NO: 1.Furthermore, the secreted eIF5A of the present invention includespolypeptides having an amino acid sequence of SEQ ID NO: 1, in which oneor more amino acids are deleted, substituted or added so long as thepolypeptides have the same function.

The secreted (hypusinated) eIF5A of the present invention has anapoptosis-inducing action, in particular, an action of inducingapoptosis caused by an oxidative stress. As a cultured cell systemcontaining cytosolic eIF5A did not undergo apoptosis, theapoptosis-inducing ability is specific to the secreted (particularlyhypusinated) eIF5A.

Furthermore, the apoptosis-inducing action of the secreted (hypusinated)eIF5A of the present invention involves both a caspase-dependent pathwayand a caspase-independent pathway.

As the secreted (hypusinated) eIF5A of the present invention not onlyinduces apoptosis caused by an oxidative stress, but also inducesapoptosis under normoxia, it is useful as a preventive or therapeuticagent for cancer, infiltrative diseases (for example, sarcoidosis), andthe like.

The pharmaceutical of the present invention can be prepared as a drugproduct through mixing, dissolution, granulation, tabletting,emulsification, encapsulation, lyophilization, and the like by using thesecreted (hypusinated) eIF5A in combination with pharmaceuticallyacceptable carriers known in the art.

For oral administration, the secreted (hypusinated) eIF5A can beprepared in dosage forms such as a tablet, a pill, a sugar-coated agent,a soft capsule, a hard capsule, a solution, a suspension, an emulsion, agel, a syrup, and a slurry in combination with pharmaceuticallyacceptable solvents, diluents, binders, stabilizers, dispersing agents,and the like.

For parenteral administration, the secreted (hypusinated) eIF5A can beprepared in dosage forms such as a solution for injection, a suspension,an emulsion, a cream, an ointment, an inhalant, and a suppository incombination with pharmaceutically acceptable solvents, diluents,binders, stabilizers, dispersing agents, and the like. In theformulation for injection, the secreted (hypusinated) eIF5A can bedissolved in an aqueous solution, preferably a physiologicallycompatible buffer such as Hanks' solution, Ringer's solution, or aphysiological saline buffer. Furthermore, a composition can have formssuch as suspension, solution, and emulsion in an oily or aqueousvehicle. Alternatively, a therapeutic agent may be produced in a form ofpowder, which is to be prepared as an aqueous solution or a suspensionbefore use using sterilized water or the like. For administration byinhalation, the secreted (hypusinated) eIF5A can be powdered incombination with a suitable base material such as lactose or starch, tothereby produce a powder mixture. For the formulation of suppository,the secreted (hypusinated) eIF5A can be mixed with a commonly usedsuppository base material such as cocoa butter. Furthermore, thetherapeutic agent of the present invention can be formulated as asustained release preparation by encapsulating the secreted(hypusinated) eIF5A in a polymer matrix or the like.

The dose and the number of doses vary depending on the dosage form, theadministration route, and the patient's symptom, age, and body weight.Generally, the dose of the secreted (hypusinated) eIF5A is in the rangeof approx. 0.001 to 1000 mg, preferably approx. 0.01 to 10 mg, per kgbody weight per day and can be administered as one dose or divided intoseveral doses per day.

As the secreted (hypusinated) eIF5A of the present invention is secretedfrom cells under an oxidative stress, the secreted (hypusinated) eIF5Acan be used for the diagnosis or determination of oxidative stress.Specifically, the concentration of the secreted (hypusinated) eIF5A in abody fluid or tissue specimen is measured. If the concentration ishigher than the concentration under a condition with no oxidativestress, the subject of the body fluid or tissue specimen can bediagnosed as being under an oxidative stress. Furthermore, if thesubject is in a treatment for a disease caused by an oxidative stress,whether the course of the treatment is favorable or not can bedetermined by measuring the concentration of the secreted (hypusinated)eIF5A.

As the secreted (hypusinated) eIF5A, the target of the measurement ofthe present invention is secreted from cells by an oxidative stress, aspecimen is preferably a body fluid and particularly preferably blood,serum, or plasma.

As an embodiment of the diagnosis or the determination of the presentinvention, immunoassay can be performed by using blood, serum, or plasmaobtained from a subject as a sample. Examples of the immunoassay includeradioimmunoassay, enzyme immunoassay, fluorescence immunoassay,luminescence immunoassay, immunoprecipitation, immunonephelometry,Western blotting, and immunodiffusion. Enzyme immunoassay is preferred,and enzyme-linked immunosorbent assay (ELISA) (for example, sandwichELISA) is particularly preferred. The above-mentioned immunoassays, suchas ELISA, can be performed by methods known to those skilled in the art.

As an example of the method for diagnosing an oxidative stress usingblood, serum, or plasma as a specimen, an anti-secreted (hypusinated)eIF5A antibody (hereinafter referred to simply as anti-eIF5A antibody)is immobilized on a support, a test sample is added thereto, andincubation is performed to allow the anti-eIF5A antibody to bind to theprotein. After that, the support is washed, and the secreted(hypusinated) eIF5A protein which binds to the support is detected viathe anti-eIF5A antibody.

Examples of the support used for the immobilization of the anti-eIF5Aantibody in the present invention include insoluble polysaccharides suchas agarose and cellulose, synthetic resins such as silicon resin,polystyrene resin, polyacrylamide resin, nylon resin, and polycarbonateresin, and insoluble supports such as glass and ferrite. These supportscan be used in forms such as beads and plates. Beads can be used byfilling them in a column or the like. As plates, multiwell plates(96-well multiwell plate etc.), biosensor chips, and the like can beused. Binding of an anti-eIF5A antibody and a support can be performedby usual methods such as chemical bonding and physical adsorption. Asall these supports, commercially available ones can be used.

Binding of an anti-eIF5A antibody and the secreted (hypusinated) eIF5Aprotein in a sample is usually performed in a buffer. Examples of thebuffer include phosphate buffer, Tris buffer, citrate buffer, boratebuffer, and carbonate buffer, and the usual range of pH is sufficient.Furthermore, the incubation is performed under conditions which havebeen commonly used, for example, incubation at 4° C. to 37° C. for oneto 24 hours. For washing after incubation, any washes can be used solong as the washes do not interfere with binding of the anti-eIF5Aantibody and the secreted (hypusinated) eIF5A protein, and examples ofthe wash include buffers including surfactants such as Tween-20.

In the method of detecting a secreted (hypusinated) eIF5A protein by thepresent invention, a control sample may be established in addition to atest sample in which the secreted (hypusinated) eIF5A protein is to bedetected. Examples of the control sample include negative controlsamples which do not contain the secreted (hypusinated) eIF5A protein,and positive control samples which contain the secreted (hypusinated)eIF5A protein. In this case, the secreted (hypusinated) eIF5A protein inthe test sample can be detected by comparing a result obtained using anegative control sample which does not contain the secreted(hypusinated) eIF5A protein, with a result obtained by using a positivecontrol sample which contains the secreted (hypusinated) eIF5A protein.Furthermore, the secreted (hypusinated) eIF5A protein contained in atest sample can be quantitatively detected by preparing a series ofcontrol samples with concentrations changed stepwise, obtaining adetection result for each control sample as a numerical value, creatinga standard curve, and quantifying by using the numerical value of thetest sample based on the standard curve.

As a preferred embodiment of detection of a secreted (hypusinated) eIF5Aprotein which binds to a support via an anti-eIF5A antibody, a methodusing an anti-eIF5A antibody labeled a labeling substance can bementioned. For example, a test sample is brought into contact with ananti-eIF5A antibody immobilized on a support, the support is washed, andthen a secreted (hypusinated) eIF5A protein is detected using a labeledantibody which specifically recognizes the secreted (hypusinated) eIF5Aprotein.

The anti-eIF5A antibody can be labeled by commonly known methods. Aslabeling substances, labeling substances known to those skilled in theart, such as fluorescent dyes, enzymes, coenzymes, chemiluminescentsubstances, and radioactive substances, can be used. Specific examplesof the labeling substances include radioisotopes (³²P, ¹⁴C, ¹²⁵I, ³H,¹³¹I, etc.), fluorescein, rhodamine, dansyl chloride, umbelliferone,luciferase, peroxidase, alkaline phosphatase, β-galactosidase,β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme,saccharide oxidase, microperoxidase, biotin, and ruthenium. When biotinis used as a labeling substance, a biotin-labeled antibody is added andthen streptavidin binding to an enzyme such as peroxidase is preferablyadded. To allow a labeling substance to bind to an anti-eIF5A antibody,known methods such as glutaraldehyde methods, maleimide methods, pyridyldisulfide methods, and periodic acid methods can be used.

Specifically, a solution containing an anti-eIF5A antibody is added to asupport such as a plate or beads to immobilize the anti-eIF5A antibodyon the support. The plate or beads are washed, and then blocking isperformed using, for example, BSA, gelatin, or albumin to preventnonspecific binding of the protein. The support is washed again, and thetest sample is added to the plate or the beads. After incubation, thesupport is washed, and a labeled anti-eIF5A antibody is added. Afterappropriate incubation, the plate or the beads are washed, and thelabeled anti-eIF5A antibody which remains on the support is detected.Detection can be performed by methods known to those skilled in the art.For example, when a radioactive substance is used for labeling,detection can be performed by liquid scintillation or RIA method. Whenan enzyme is used for labeling, a substrate is added and detection canbe performed through an enzymatic change of the substrate, for example,coloration can be detected with an absorption spectrometer. Specificexamples of the substrate include2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt(ABTS), 1,2-phenylenediamine(ortho-phenylenediamine), and3,3′,5,5′-tetramethylbenzidine (TMB). When a fluorescent substance or achemiluminescent substance is used, it can be detected with a detectionluminometer.

As a particularly preferred embodiment of the method for detecting asecreted (hypusinated) eIF5A protein of the present invention, a methodusing a biotin-labeled anti-eIF5A antibody and streptavidin can bementioned.

Specifically, a solution containing an anti-eIF5A antibody is added to asupport such as a plate to immobilize the anti-eIF5A antibody thereon.The plate is washed, and then blocking is performed using BSA or thelike to prevent nonspecific binding of the protein. The plate is washedagain, and a test sample is added to the plate. After incubation, theplate is washed, and then a biotin-labeled anti-eIF5A antibody is added.After suitable incubation, the plate is washed, avidin which binds to anenzyme such as alkaline phosphatase or peroxidase is added. Afterincubation, the plate is washed, a substrate for the enzyme which bindsto avidin is added, and the secreted (hypusinated) eIF5A protein isdetected by using an enzymatic change of the substrate or the like as anindicator.

As another embodiment of the method for detecting a secreted(hypusinated) eIF5A protein of the present invention, a method using oneor more primary antibodies which specifically recognize a secreted(hypusinated) eIF5A protein and one or more secondary antibodies whichspecifically recognize the primary antibodies can be mentioned.

For example, a test sample is brought into contact with one or moreanti-eIF5A antibodies immobilized on a support and incubated, then thesupport is washed, and, after wash, a secreted (hypusinated) eIF5Aprotein bound is detected by using primary anti-eIF5A antibodies and oneor more secondary antibodies which specifically recognize the primaryantibodies. In this case, the secondary antibodies are preferablylabeled with a labeling substance.

As another embodiment of the method for detecting a secreted(hypusinated) eIF5A protein of the present invention, a method using anagglutination reaction can be mentioned. In this method, a secreted(hypusinated) eIF5A protein can be detected by using an anti-eIF5Aantibody-sensitized carrier. Any antibody-sensitized carriers can beused so long as the carriers are insoluble, do not cause a nonspecificbinding reaction, and are stable. Examples of the antibody-sensitizedcarrier include latex particles, bentonite, collodione, kaolin, andimmobilized sheep erythrocytes, and latex particles are preferably used.Examples of the latex particles include polystyrene latex particles,styrene-butadiene copolymer latex particles, and polyvinyltoluene latexparticles, and polystyrene latex particles are preferably used.Sensitized particles are mixed with a sample, and the mixture is stirredfor a predetermined time. As the degree of agglutination of particles isgreater when a higher concentration of a secreted (hypusinated) eIF5Aprotein is contained in the sample, the secreted (hypusinated) eIF5Aprotein can be detected by observing agglutination macroscopically.Furthermore, the secreted (hypusinated) eIF5A protein can also bedetected by measuring turbidity due to agglutination with aspectrophotometer or the like.

As another embodiment of the method for detecting the protein of thepresent invention, for example, a method using a biosensor whichutilizes a surface plasmon resonance phenomenon can be mentioned. Abiosensor utilizing a surface plasmon resonance phenomenon enablesobservation of a protein-protein interaction in real time as a surfaceplasmon resonance signal by using a trace amount of proteins withoutlabeling. For example, each binding of an anti-eIF5A antibody and asecreted (hypusinated) eIF5A protein can be detected by using abiosensor such as BIAcore (Biacore International AB). Specifically, atest sample is brought into contact with a sensor chip on which ananti-eIF5A antibody is immobilized, each secreted (hypusinated) eIF5Aprotein binding to the anti-eIF5A antibody can be detected as a changein the resonance signal.

The detection method of the present invention can also be automatedusing various automatic test apparatus, and thus a large amount ofsamples can be tested at a time.

The agent for diagnosing an oxidative stress of the present inventionmay be provided in the form of a kit. The diagnostic agent for anoxidative stress of the present invention contains at least ananti-eIF5A antibody. When the diagnostic agent is based on EIA such asELISA, a carrier which solidifies an antibody may be contained, or anantibody may be allowed to bind to a carrier beforehand. When thediagnostic agent is based on agglutination using a carrier such aslatex, a carrier to which an antibody is adsorbed may be contained.Furthermore, the diagnostic agent may suitably contain a blockingsolution, a reaction solution, a reaction terminating solution, areagent for treating a sample, and the like.

The anti-eIF5A antibody used in the diagnostic agent of the presentinvention may be one binding specifically to each secreted (hypusinated)eIF5A protein, regardless of the origin, the type (monoclonal orpolyclonal), and the shape of the anti-eIF5A antibody. Specifically,known antibodies such as mouse antibodies, rat antibodies, avianantibodies, human antibodies, chimera antibodies, and humanizedantibodies can be used. Antibodies may be polyclonal antibodies, butmonoclonal antibodies are preferred. Commercially available antibodiesmay be used so long as the antibodies can be measured specifically withhigh sensitivity.

Furthermore, an anti-eIF5A antibody immobilized on a support and ananti-eIF5A antibody labeled with a labeling substance may recognize thesame epitope of a secreted (hypusinated) eIF5A protein, but differentepitopes are preferably recognized, and sites are not particularlylimited.

Anti-eIF5A antibodies used in the present invention can be obtainedusing known means as polyclonal or monoclonal antibodies. As theanti-eIF5A antibodies used in the present invention, mammal-derived oravian-derived monoclonal antibodies are preferred. Mammal-derivedmonoclonal antibodies are particularly preferred. Mammal-derivedmonoclonal antibodies include antibodies produced by using hybridoma andantibodies produced by using a host transfected with an expressionvector having an antibody gene through genetic engineering technique.

Monoclonal antibody-producing hybridoma can be prepared basically byusing known techniques as follows. Specifically, a secreted(hypusinated) eIF5A protein is used as a sensitizing antigen andimmunized by using the antigen according to a usual immunologicalmethod, the obtained immunocyte is fused with a known parent cell by ausual cell fusion method, and screening a monoclonal antibody-producingcell by a usual screening method, to thereby obtain the hybridoma.

Specifically, a monoclonal antibody can be prepared as follows.

A purified secreted (hypusinated) eIF5A protein is used as a sensitizingantigen. Alternatively, a partial peptide of the secreted (hypusinated)eIF5A protein can also be used as a sensitizing antigen. At this time,the partial peptide can be obtained based on the amino acid sequence ofa human secreted (hypusinated) eIF5A protein by chemical synthesis, byincorporating a part of the human eIF5A gene into an expression vector,or by degrading a natural human secreted (hypusinated) eIF5A protein bya proteolytic enzyme. The portion and the size of the human secreted(hypusinated) eIF5A protein used as a partial peptide are not limited.

Mammals immunized with a sensitizing antigen are not particularlylimited, but are preferably selected in view of the compatibility with aparent cell used for cell fusion. Generally, rodents such as mice, ratsand hamsters, birds, rabbits, monkeys, and the like are used.

Animals are immunized with a sensitizing antigen according to knownmethods. For example, as a common method, a sensitizing antigen isinjected to mammals intraperitoneally or subcutaneously. Specifically, asensitizing antigen is diluted with PBS (phosphate-buffered saline),physiological saline, or the like and suspended in an appropriatevolume, mixed with an appropriate amount of a usual adjuvant such asFreund's complete adjuvant, as required, emulsified, and thenadministered to mammals several times every 4 to 21 days. Furthermore,an appropriate carrier can also be used at the time of immunization witha sensitizing antigen. In particular, when a partial peptide with a lowmolecular weight is used as a sensitizing antigen, the partial peptideis preferably allowed to bind to a carrier protein such as albumin orkeyhole limpet hemocyanin and used for immunization.

After a mammal is thus immunized, and the elevation of the level of arequired antibody in serum is confirmed, immunocytes are collected fromthe mammal and subjected to cell fusion. Particularly preferred examplesof immunocytes include splenic cells.

As the other parent cell fused with the immunocyte, a mammal myelomacell is used. Preferred examples of the myeloma cell include variousknown cell strains, for example, P3 (P3×63Ag8.653) (J. Immnol. [1979]123, 1548-1550), P3×63Ag8U.1 (Current Topics in Microbiology andImmunology [1978] 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J.Immunol. [1976] 6, 511-519), MPC-11 (Margulies. D. H. et al., Cell[1976] 8, 405-415), SP2/0 (Shulman, M. et al., Nature [1978] 276,269-270), FO (de St. Groth, S. F. et al., J. Immunol. Methods [1980] 35,1-21), 5194 (Trowbridge, I. S. J. Exp. Med. [1978] 148, 313-323), and8210 (Galfre, G. et al., Nature [1979] 277, 131-133).

The immunocytes and myeloma cells can be fused basically by knownmethods, for example, the method of Kohler, Milstein, et al. (Kohler. G.and Milstein, C., Methods Enzymol. [1981] 73, 3-46).

More specifically, the cell fusion is carried out in a usual nutrientculture broth in the presence of, for example, a cell fusion promoter.Examples of the fusion promoter include polyethylene glycol (PEG) andSendai virus (HVJ). To increase fusion efficiency as required, aids suchas dimethyl sulfoxide can be further added for use.

The ratio of an immunocyte and a myeloma cell used can be arbitrarilyselected. For example, the ratio of an immunocyte to a myeloma cell ispreferably 1 to 10. Examples of the culture broth used for the cellfusion include RPMI 1640 culture broth suitable for proliferation of themyeloma cell strains, MEM culture broth, and other usual culture brothsused for this type of cell culture. Serum replacement fluids such asfetal calf serum (FCS) can also be used in combination.

For cell fusion, a target fused cell (hybridoma) is formed by mixingpredetermined amounts of the immunocyte and myeloma cell well in theculture broth, adding PEG solution (for example, an average molecularweight of approx. 1000 to 6000) heated at approx. 37° C. beforehandusually at concentration a of 30 to 60% (w/v), and mixing well.Subsequently, an appropriate culture broth is serially added, and themixture is centrifuged to remove the supernatant. By repeating thisprocedure, cell fusing agents or the like which are undesirable forproliferation of hybridoma and the like are removed.

The hybridoma thus obtained is selected by culturing the cells in ausual selective culture broth, for example, HAT culture broth (culturebroth containing hypoxanthine, aminopterin, and thymidine). The culturein the HAT culture broth is continued for a sufficient time (normally,several days to several weeks) to kill cells (non-fused cells) otherthan the target hybridoma. Subsequently, hybridoma which produces atarget antibody is subjected to screening and single cloning by a usuallimiting dilution method.

Screening and single cloning of the target antibody can be carried outby a screening method based on known antigen-antibody reactions. Forexample, an antigen is allowed to bind to a carrier such as beads madeof polystyrene or the like or a commercially available 96-wellmicrotiter plate and reacted with the culture supernatant of hybridoma,the carrier is washed, and then whether or not a target antibody whichreacts with the sensitizing antigen in the culture supernatant iscontained can be determined by allowing an enzyme-labeled secondaryantibody or the like to react. The hybridoma which produces the targetantibody can be cloned by a limiting dilution method or the like. Atthis time, the antigen used for immunization can be used as the antigen.

The hybridoma producing a monoclonal antibody which is thus prepared canbe subcultured in a usual culture broth and stored in liquid nitrogenfor a long period.

To obtain a monoclonal antibody from the hybridoma, a method ofculturing the hybridoma according to a usual method and obtaining as theculture supernatant or a method of administering the hybridoma to acompatible mammal to proliferate it and obtaining the antibody asascites can be used. The former method is suitable to obtain high-purityantibodies, while the latter method is suitable for mass production ofantibodies.

Antibodies used in the present invention are not limited to the wholeantibody molecules and may be fragments or modified fragments of anantibody so long as the antibodies bind to the secreted (hypusinated)eIF5A protein. Both bivalent antibodies and univalent antibodies arealso included. Examples of the fragments of an antibody include Fab,F(ab′)2, Fv, Fab/c containing one Fab and complete Fc, and single-chainFv (scFv) to which Fv of the H chain or the L chain is linked with anappropriate linker. Specifically, an antibody is treated with an enzymesuch as papain or pepsin to produce an antibody fragment, or the genecoding for the antibody fragment is constructed, and the gene isintroduced into an expression vector and expressed in an appropriatehost cell (for example, refer to Co, M. S. et al., J. Immunol. [1994]152, 2968-2976; Better, M. & Horwitz, A. H., Methods in Enzymology[1989] 178, 476-496, Academic Press, Inc.; Plueckthun, A. & Skerra, A.,Methods in Enzymology [1989] 178, 476-496, Academic Press, Inc.; Lamoyi,E., Methods in Enzymology [1989] 121, 652-663; Rousseaux, J. et al.,Methods in Enzymology [1989] 121, 663-669; and Bird, R. E. et al.,TIBTECH [1991] 9, 132-137).

scFv can be obtained by linking the V region of H chain and the V regionof light (L) chain of an antibody. In this scFv, the V region of H chainand the V region of L chain are linked with a linker, preferably with apeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A.[1988] 85, 5879-5883). The V region of H chain and the V region of Lchain in scFv may be derived from any antibodies described in thepresent specification. Examples of the peptide linker for linking the Vregions include arbitrary single-stranded peptides including 12 to 19amino acid residues.

DNA coding for scFv can be obtained by PCR amplification by using, as atemplate, a whole sequence of DNA coding for the H chain or the V regionof H chain and DNA coding for the L chain or the V region of L chain inthe antibody, or a DNA portion coding for a required amino acid sequenceand a primer pair which defines the both ends of the DNA, and furtheramplification by using DNA coding for a peptide linker portion and aprimer pair which defines the linker so that the both ends should belinked to the H chain and the L chain.

Furthermore, once DNA coding for scFv is prepared, an expression vectorcontaining the DNA and a host transformed with the expression vector canbe obtained by a usual method. scFv can be obtained by a usual methodusing the host.

Fragments of the antibody can be expressed by using the obtained genesas described above and produced by the host. The “antibodies” in thepresent invention include the antibody fragments.

As modified antibodies, anti-eIF5A antibodies which bind to variousmolecules such as labeling substances can also be used. The “antibodies”in the present invention also include the modified antibodies. Themodified antibodies can be obtained by subjecting the obtainedantibodies to chemical modification. It is noted that methods formodifying antibodies have already been established in the art.

Furthermore, antibodies used in the present invention may be bispecificantibodies. Bispecific antibodies may be bispecific antibodies havingantigen-binding sites which recognize different epitopes on a molecule,or one having one antigen-binding site which may recognize a secreted(hypusinated) eIF5A protein and the other antigen-binding site which mayrecognize a labeling substance or the like. The bispecific antibody canbe prepared by linking pairs of the H regions and the L regions of twoantibodies or obtained by fusing hybridomas producing differentmonoclonal antibodies to prepare a fusion cell which produces abispecific antibody. Furthermore, bispecific antibodies can also beprepared by genetic engineering techniques.

As the secreted (hypusinated) eIF5A of the present invention inducesapoptosis caused by an oxidative stress, an agent for inhibiting thesecreted (hypusinated) eIF5A can be used as an apoptosis inhibitor.Examples of the secreted (hypusinated) eIF5A inhibitor includeanti-eIF5A neutralizing antibodies. The origin, type (monoclonal orpolyclonal) and the shape of anti-eIF5A neutralizing antibodies are notlimited. Specific examples of the anti-eIF5A neutralizing antibodiesinclude known antibodies such as mouse antibodies, rat antibodies, avianantibodies, human antibodies, chimera antibodies, and humanizedantibodies. Antibodies may be polyclonal antibodies, but monoclonalantibodies, which can be mass-produced, are preferred in view ofadministration into a living body. It is sufficient that the secreted(hypusinated) eIF5A can be neutralized in the living body to inhibit theaction of the secreted (hypusinated) eIF5A.

Here, as the apoptosis inhibitor of present invention inhibits apoptosiscaused by an oxidative stress, the apoptosis inhibitor of presentinvention is useful as an preventive or therapeutic agent for variousdiseases caused by an oxidative stress, such as, for example, not onlyischemia-reperfusion injury and ultraviolet ray/radiation damage, butalso atherosclerosis, aging, and the like, which are accelerated by anoxidative stress. Examples of “ischemic” diseases include ischemic heartdiseases (angina pectoris and acute myocardial infarction), cerebralinfarction, lung thromboembolism, ischemic intestinal diseases (acutemesenteric arterial occlusion and ischemic colitis), renal embolism, andcases of heart arrest in heart transplantation, open-heart surgery, andthe like using an artificial heart-lung apparatus.

The pharmaceutical of the present invention can be prepared as a drugproduct by mixing, dissolution, granulation, tableting, emulsification,encapsulation, lyophilization, or the like of a secreted (hypusinated)eIF5A inhibitor with a pharmaceutically acceptable carrier known in thistechnical field.

For oral administration, the secreted (hypusinated) eIF5A inhibitor canbe prepared in dosage forms such as a tablet, a pill, a sugar-coatedagent, a soft capsule, a hard capsule, a solution, a suspension, anemulsion, a gel, a syrup, and a slurry with pharmaceutically acceptablesolvents, diluents, binders, stabilizers, dispersing agents, and thelike.

For parenteral administration, the secreted (hypusinated) eIF5Ainhibitors can be prepared as a drug product in dosage forms such as asolution for injection, a suspension, an emulsion, a cream, an ointment,an inhalant, and a suppository in combination with pharmaceuticallyacceptable solvents, diluents, binders, stabilizers, dispersing agents,and the like. In the formulation for injection, the secreted hypusinatedeIF5A inhibitor can be dissolved in an aqueous solution, preferably in aphysiologically compatible buffer such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. Furthermore, a composition canbe in forms such as suspension, solution, or emulsion in an oily oraqueous vehicle. Alternatively, a therapeutic agent may be produced in aform of powder, and an aqueous solution or a suspension may be preparedwith sterilized water before use. For administration by inhalation, thesecreted hypusinated eIF5A inhibitor is powdered, and a powder mixturecan be prepared with a suitable base material such as lactose or starch.Suppository can be produced by mixing the secreted (hypusinated) eIF5Ainhibitor with a commonly used suppository base material such as cocoabutter. Furthermore, the therapeutic agent of the present invention canbe encapsulated in a polymer matrix or the like and formulated as asustained release preparation.

The dose and the number of doses vary depending on the dosage form, theadministration route, and the patient's symptom, age, and body weight.Generally, the secreted (hypusinated) eIF5A inhibitor in the range ofapprox. 0.001 to 1000 mg, preferably approx. 0.01 to 10 mg, per kg bodyweight per day, can be administered as one dose or the dose can bedivided into several times per day. As acute phase treatment of acutemyocardial infarction, cerebral infarction, and the like, reperfusiontherapy for reopening the occluded vessel using a thrombolytic agent(urokinase, tissue plasminogen activation factor [tPA], etc.) or aballoon catheter is generally performed in the early phase of thedisease. The anti-(secreted) eIF5A neutralizing antibody can beintravenously (as one shot) administered before or during reperfusion(if possible, 5 to 10 min before). When an occluded vessel is expandedwith a balloon catheter and a stent is inserted, the anti-(secreted)eIF5A neutralizing antibody can be similarly administered before orduring reperfusion (vascular dilatation). In a drug delivery ballooncatheter, the anti-(secreted) eIF5A neutralizing antibody is preferablyadministered before or during blood reflow by vascular dilatation.

EXAMPLES

The present invention will be described in detail with reference to thefollowing examples. However, the scope of the present invention is notlimited to these examples.

First, the experimental methods will be described.

(Cells Culture)

According to the description in Circ. Res. 78, 82-90 (1996), a primaryculture of ventricular cardiac myocytes was prepared from neonatal rats.These cells were cultured for 2 days until they were confluent.

(Hypoxia and Reoxygenation)

According to the description in Circ. Res. 78, 82-90 (1996), a hypoxiacondition (N₂ 95%, CO₂ 5%, and O₂<0.1%) was achieved. After incubatingin a hypoxic condition for 60 min (for cardiac myocytes) or 20 min (forquail muscle cells), the cells were reoxygenated by immediatelyreplacing the hypoxic PBS with normoxic PBS for 10 min. The supernatantPBS was collected after 10 min of reoxygenation asreoxygenation-conditioned PBS (RCP). Also the supernatant PBS wascollected after 10 min of incubation with non-stimulated cardiacmyocytes under normoxia as control-conditioned PBS (CCP). The RCP andCCP were concentrated and the fractions of molecular weight>10 kD werecollected by using centripreps (YM-10; Millipore Corporation).

(Chromatofocusing)

Both RCP and CCP were collected from the same number of cells, loadedonto a MONO-P (5 mm×200 mm) column (GE Healthcare) connected to a GILSONHPLC system at a flow rate of 1 ml min⁻¹, equilibrated with Solution A(0.025M BisTris, pH 7.1), eluted with Solution B (10% Poly Buffer74,pH4.0), and washed out the column-bound components with Solution C (1MNaCl/10% Poly Buffer74, pH 4.0).

(Cloning and Plasmid Construction)

Human eIF5A cDNA was amplified by RT-PCR from total RNA isolated fromSaSO2 cells (human osteoblast-like cell line) and subcloned into theEcoRI/Xho I site of pcDNA4/myc-His Vector (Invitrogen). Then, FLAG plusHis-tagged eIF5A construct was generated with the following primers.

The forward primer was

(SEQ ID NO: 2) 5′-CACCGAATTCAAAATGGCAGATGACT-3′,and the reverse primer was

(SEQ ID NO: 3) 5′-ATATACTCGAGTCAGTGATGGTGATGGTGGTGCTTGTCATCGTCGTCCTTGTAA TCTTTTGCCATGGCCTTGATTG-3′

Then the construct was subcloned into pcDNA3.1 Directional TOPO Vector(Invitrogen).

(Recombinant eIF5A Protein)

Flag plus His-tagged eIF5A and mutant eIF5A (K50A) were transientlyexpressed in a quail muscle cell line (clone CRL-1962, ATCC) with FuGENEHD Transfection Reagent (Roche). After 48 hours of transfection, thecells were subjected to hypoxia for 20 min followed by reoxygenation for10 min. The reoxygenated PBS were collected and concentrated, and thenthe recombinant proteins were purified with Ni-NTA Purification System(Invitrogen; according to the manufacture's instruction). Cytosolicrecombinant eIF5A was also collected from transfected cells under anative condition (normoxia).

(Polyclonal Anti-eIF5A Antibody)

Rabbit anti-eIF5A polyclonal antibodies (J-M) and (J-C) were generatedagainst human eIF5A peptide (amino acid residues 38 to 57, which includethe hypusination site, and amino acid residues 138 to 154, respectively;coupled to keyhole limpet hemocyanin).

(Western Blot Analysis of Cultured Cardiac Myocytes)

Cultured cardiac myocytes were treated with the recombinant eIF5A (RCP)for 5 min, the medium was immediately aspirated, and the cells werefrozen with liquid nitrogen. As described elsewhere, the cells werelysed with buffer A on ice, and the cell lysate was centrifuged. Thesupernatant was suspended in Lemli's sample buffer. A portion of thesample was subjected to Western blot analysis by using rabbit polyclonalphosphospecific anti-ERK1/2 (Thr202/Tyr204) antibody (Cell Signaling,Inc.). Another portion of the same sample was subjected to Western blotanalysis by using rabbit polyclonal control anti-ERK1/2 antibody (CellSignaling, Inc.). The antibody-antigen complex was colored by achemiluminescence system using alkaline phosphatase (New EnglandBiolabs, Inc.).

(Immunofluorescence)

Immunofluorescence staining of cultured cardiac myocytes and myocardialtissue were performed by Tyramide Signal Amplification (TSA) technologyfor fluorescence (TSA™ Biotin System, NEN Life Science Products,PerkinElmer; according to the manufacture's instruction). Doubleimmunostaining for cardiac myosin was performed according to the sameprocedure as described in Biochem. Biophys. Res. Commun. 317, 162-168(2004).

(Immunoelectron Microscopy)

Cultured cardiac myocytes were subjected to hypoxic condition for 60 minfollowed by reoxygenation for 10 min, fixed in 4% paraformaldehyde for 2hours, washed with PBS, and dehydrated in a graded series (50 to 100%)of cold ethanol. The cells were embedded in LR White Resin (Nisshin EM,Co. Ltd., Japan)/100% ethanol (1:1) for 2 hours, then embedded in pureLR White Resin, and polymerized under ultraviolet light irradiation at4° C. overnight. Ultrathin sections were prepared, blocked with 1%bovine serum albumin in PBS and incubated with anti-eIF5A antibody (J-M)overnight. The section was washed in PBS, incubated with Protein Aconjugated gold colloidal particles-20 nm (EY Laboratories, Inc.) andthen examined with an electron microscope (H-7000, HITACHI, Japan).

(Electron Microscopy)

The cardiac myocytes were treated with recombinant eIF5A for apredetermined period. The cells were fixed in 2% glutaraldehyde,postfixed in 2% osmium tetraoxide, dehydrated in ethanol, and embeddedin a resin. Ultrathin section were prepared, stained with uranyl acetateand lead citrate, and examined with an electron microscope (H-7000,HITACHI, Japan).

(Terminal Deoxynucleotidyl Transferase Nick-End Labeling (TUNEL)Staining)

TUNEL staining was performed with In Situ Apoptosis Detection Kit(TAKARA BIO Inc., Shiga, Japan; according to the manufacture'sinstruction), and then a diaminobenzidine (DAB) reaction was carriedout. To distinguish cardiac myocytes from non-muscle cells, cells wereincubated with mouse anti-cardiac myosin mAb (clone CMA19) and alkalinephosphatase-labeled anti-mouse IgG antibody (Santa Cruz Biotechnology,Inc.) and then reacted with a substrate (alkaline phosphatase substratekit III, Vector Laboratories, Inc.), which produces a blue reactionproduct.

(Analysis of Subcellular Localization of Apoptosis Inducing Factor (AIF)in Cultured Cardiac Myocytes)

Tyramide Signal Amplification (TSA) technology was used for fluorescence(TSA™ Biotin System, NEN Life Science Products, PerkinElmer; accordingto the manufacture's instruction). Cardiac myocytes were treated with arecombinant eIF5A (RCP) for a predetermined time and fixed with 4%paraformaldehyde in PBS for 15 min. The cells were washed with PBS andthen incubated with rabbit anti-AIF mAb (E20; Epitomics Inc.) for 1hour. The cells were washed, then incubated with biotinylatedanti-rabbit IgG antibody (Chemicon International, Inc.) for 1 hour,washed, and incubated with streptavidin-horseradish peroxidase (Vector)for 30 min. The cells were washed in TNT buffer (0.1 M Tris-HCl, pH 7.5,0.15 M NaCl, 0.05% Tween 20) and then incubated withbiotinylated-tyramide for appropriate time (3 to 10 min). The cells werewashed in TNT buffer and then incubated with FITC-labeled avidin-D(Vector) for 30 min. Nuclei were stained with 1 μg·mL⁻¹ Hoechst 33342(DOJINDO Laboratories, Kumamoto, Japan). Confocal laser microscopy wasperformed on LSM510 Laser Scanning Microscope (Zeiss).

(Analysis of Release of Cytochrome c from Mitochondria)

Cultured cardiac myocytes were treated with a recombinant eIF5A for apredetermined time. Nucleic, mitochondrial, and cytosolic fraction ofthe cells were prepared as described in Non Patent Literatures 1 and 2.Briefly, 3×10⁶ cells were washed in PBS, and suspended in 250 μl of alysis buffer (250 mM sucrose, 50 mM Pipes/KOH, pH7.4, 50 mM KCl, 5 mMEGTA, 2 mM MgCl₂, 1 mM DTT, and 1 mM PMSF). After 30 min on ice, thecells were lysed with 40 strokes using pestle B in a Dounce homogenizer,and centrifuged at 80 g for 10 min. The pellets were collected as thenucleic fraction. Then, the supernatants were prepared from cell lysatesby centrifugation at 20,000 g for 20 min. The pellets were collected asthe mitochondrial fraction, and the supernatants were regarded as thecytosolic fraction. The nucleic, mitochondrial, and cytosolic fractionswere subjected to Western blot analysis with rabbit anti-AIF mAb (E20;Epitomics Inc., CA, USA) or mouse anti-cytochrome c mAb (7H8.2C12; LabVision Corp., CA, USA). The antibody-antigen complexes were developedwith a chemiluminescence system using alkalinephosphatase (New EnglandBiolabs, Inc.).

(Annexin V-Staining of Cultured Cardiac Myocytes)

Cardiac myocytes were treated with a recombinant eIF5A (RCP) for apredetermined time. The cells were incubated with biotinylated-annexin Vin 1× binding buffer (Annexin V-Biotin Apoptosis Detection Kit,BioVision Inc.) for 5 min and then fixed with 2% paraformaldehyde in PBSfor 15 min. After washing in PBS, the cells were incubated withstreptavidin-horseradish peroxidase (Vector) for 30 min. The subsequentprocedures for immunofluorescence by TSA technology were the same as forAIF. To distinguish cardiac myocytes from non-muscle cells, cells werefurther incubated with mouse anti-cardiac myosin (clone CMA19) mAb (NonPatent Literature 1), followed by incubation with tetramethyl rhodamineisothiocyanate (TRITC)-conjugated anti-mouse IgG antibody, and thenphotographed in a fluorescence microscope.

(Ischemia and Reperfusion)

Rats (male, 250 to 280 g) were subjected to coronary artery ligationaccording to J. Pathol. 180, 305-310 (1996). Briefly, rats wereanesthetized with sodium pentobarbital (40 mg Kg⁻¹, intraperitoneally),intubated, and ventilated with room air (tidal volume, 20 ml/Kg, 60 min)with a respirator (SN-480-7, Shinano Manufacturing Co., Ltd., Tokyo,Japan). After lateral thoracotomy and pericardiectomy, a 6-0 silk stitchwas placed near the intramyocardial location of the left coronary arterybeneath the left atrial appendage. Coronary artery occlusion wasperformed by pressing a short length of tube over the ends of the sutureand clamping it firmly against the heart. Reperfusion was achieved byremoving the clamp. The standard limb lead II electrocardiogram wasmonitored continuously. The ischemia and reperfusion of the regionalmyocardium were confirmed by following the changes of the ST segmentlevel on the electrocardiogram and observing the change in the color ofthe myocardium.

(Immunohistochemistry)

Rats were slaughtered at each of time point after myocardialischemia/reperfusion. Cryostat sections (6-μm thick) of heart ventriclewere prepared, air-dried, and fixed in acetone for 5 min. The sectionswere incubated with rabbit polyclonal anti-eIF5A antibody (J-M) at 37°C. for 1 hour, and then incubated with biotinylated anti-rabbit IgGantibody (Vector Laboratories, Inc., CA) at 37° C. for 1 hour. Thesubsequent procedures by TSA technology for immunofluorescence weresimilar to the procedures for annexin V.

(Immunocytochemistry)

To distinguish cardiac myocytes from non-muscle cells, double-stainingwas performed for cardiac myosin and eIF5A with mouse anti-cardiacmyosin (CMA19) mAb followed by incubation with TRITC-conjugatedanti-mouse IgG antibody as for Annexin V. The procedure for staining ofeIF5A was the same as the procedure for tissue samples.

(Two-Dimensional Electrophoresis of myc- and FLAG-Tagged RecombinanteIF5A Protein)

Quail muscle cells were transfected with eIF5A-myc-His vector and myc-and His-tagged recombinant eIF5A protein was collected from thecytoplasm of the transfected cells. Similarly, quail muscle cells weretransfected with the eIF5A-FLAG-His vector, the cells were incubated inhypoxia/reoxygenation-conditioned PBS (RCP), and a FLAG- and His-taggedrecombinant eIF5A protein was collected from the culture supernatant.The proteins were treated with Ni-NTA Purification System (Invitrogen),and after that gel filtration was performed with phosphate buffercontaining 0.15 M NaCl (pH 7.4) using Superdex 200™ 10-300GL (GEHealthcare, 1.0×30 cm, bed capacity 24 mL) to purify the myc- andFLAG-tagged recombinant eIF5A proteins. The first dimensionalisoelectric focusing was performed with Immobiline DryStrip (pH 4 to 7,GE Healthcare) which had been allowed to swell overnight with a sampledissolved in 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM dithioerythritol[DTE], 2% IPG buffer pH 4 to 7 (GE Healthcare) and bromophenol blue[BPB]. The electrophoresis was performed at a voltage of 30 V for 7hours, at 60V for 7 hours, at 60 to 200 V for 30 min, at 200 to 500 Vfor 30 min, at 500 to 1000 V for 30 min, at 1000 to 8000 V for 30 min,and at 8000 V for 2 hours (19.4 kVh in total). The second dimensionalSDS-PAGE was performed with a gel (concentrated gel 4% acrylamide, 2.6%piperazine diacrylamide [PDA], separating gel 12% acrylamide, 2.6% PDA)and the electrophoresis was performed at 12 mA for 2 hours. The gel wastransferred to a PVDF membrane, and then the myc- and FLAG-taggedrecombinant eIF5A proteins were analyzed by Western blotting. First, theproteins were analyzed using anti-myc monoclonal antibody (Invitrogen),biotinylated anti-mouse IgG antibody and Vectastain ABC-AP Kit (Vector)and then detected with alkaline phosphatase and chemiluminescence. Then,the proteins were analyzed using anti-FLAG monoclonal antibody (M2,Sigma) and horseradish peroxidase (HRP)-labeled anti-mouse IgG antibodyand detected with Konica Immunostain HRP-1000 Kit (Konica Corporation).

(Apoptosis Induction in Various Cancer Cells by Recombinant eIF5A)

Quail muscle cells (ATCC; CRL-1962), HeLa cells, human hepatocellularcarcinoma cells (ATCC; HB-8064), and human glioblastoma cells (ATCC;CRL-1690) were incubated on a culture slide (BD Falcon) until they wereconfluent. These cells were treated with a recombinant eIF5A (10 μg/ml)for a predetermined time and fixed. Terminal deoxynucleotidyltransferase nick-end-labeled (TUNEL) staining was performed in situ withan Apoptosis Detection Kit (Takara Bio Inc.).

Example 1

(Identification of Humoral Factor Secreted fromHypoxia/Reoxygenation-Conditioned Cultured Cells)

(1) To identify an apoptosis-inducting humoral factor derived fromhypoxia/reoxygenation-conditioned media, fractions having a relativemolecular weight (M_(r)) of higher than 10 kD were collected from thesupernatant PBS of cardiac myocytes, which were subjected to hypoxia for60 min and then reoxygenation for 10 min, as reoxygenation-conditionedPBS (RCP) and concentrated.

This is because these fractions have an ERK activation activity and anapoptosis-inducing activity.

Furthermore, supernatant PBS incubated together with non-stimulatedcardiac myocytes under normoxia for 10 min was collected ascontrol-conditioned PBS (CCP) and concentrated. Proteins in RCP and CCPwere separated by chromatofocusing (FIG. 1).

(2) The ERK-activation activity of each of fractions, which seemed to beone of the most sensitive markers of the target factor, was monitoredusing cultured rat cardiac myocytes by Western blotting withphosphor-specific anti-ERK1/2 antibody (Cell Signaling Technology,Inc.). Then it was found that fractions 49 to 52 (high-salt [1M NaCl]fractions) in both RCP and CCP groups had strong activity (activity:RCP>CCP; FIG. 1), and fractions 5 to 8 (which were passed throughfractions) in RCP had a weak to moderate activity but much less thanthat of the fractions 49 to 52. Then, the most active fractions (49 to52) in each group were subjected to two-dimensional electrophoresis(FIG. 2 a: left panel, RCP; right panel, CCP). As the active componentswere not eluted by Solution B, the components are considered acidic.Among spots with low pI values, spot 1 (M_(r) 14.4 kD and pI 4.8)indicated by an arrow in RCP (FIG. 2 a, left panel) was not present inCCP (FIG. 2 a, right panel), and spot 2 (M_(r) 16.8 kD and pI 5.1)indicated by an arrow are slightly present in CCP (spot 2′). Therefore,these spots seemed to be newly appeared in response tohypoxia/reoxygenation. By LC-MS/MS analysis of protein spots 1 and 2,thioredoxin and eukaryote translation initiating factor (eIF) 5A,respectively, were identified.

Example 2

(Apoptosis-Inducing Ability of Secreted eIF5A)

(1) As thioredoxin has been known to act as antioxidant by reducingother proteins and play a protective role against oxidativestress-induced cell injury, eIF5A was considered to be a candidate of anapoptosis inducing humoral factor. eIF5A is the only protein known tocontain a unique amino acid hypusine, which is formedpost-translationally by two-steps of an enzymatic reaction. eIF5A hasbeen known to be localized mainly in the cytoplasm under normalconditions where hypusinated eIF5A facilitates translation of mRNAsinvolved in cell proliferation. However, no study to date has reportedthat eIF5A can be released extracellularly and act as anapoptosis-inducing factor. To confirm that eIF5A is actually releasedfrom cardiac myocytes in response to hypoxia/reoxygenation and thereleased eIF5A can induce apoptosis of cardiac myocytes, quail musclecells (CRL-1962, ATCC) were transfected with an expression vectorcontaining the FLAG- and His-tagged human eIF5A gene, incubated withhypoxic PBS, and then incubated with normoxic PBS. Thereoxygenation-conditioned PBS (RCP) was collected, concentrated, andpurified using Ni-NTA Purification System (Invitrogen), and recombinantprotein ([RCP] re-eIF5A) was extracted by gel filtration.

(2) Similarly, recombinant human eIF5A ([cytosolic] re-eIF5A) wasextracted from cytosolic fractions of the untreated transfected cells.

(3) After these recombinant eIF5A proteins were separated bytwo-dimensional electrophoresis, the resultant was analyzed by Westernblotting with anti-FLAG M2 mAb (Sigma) (FIG. 2 b). The blot withanti-FLAG M2 mAb showed mainly two forms of recombinant eIF5A proteinsfor both cytosolic and secreted (RCP) eIF5A (FIG. 2 b; [A, B] and [A′,B′]). Specifically, spots having higher pI values, such as hypusinatedeIF5A (spots B and B′ in FIG. 2 b), and spots having lower pI values,such as unhypusinated eIF5A (spots A and A′ in FIG. 2 b) wereidentified. There was no detectable spots of deoxyhypusinatedintermediate (references Taylor, C. A., et al. Exp. Cell Res. [2007]313, 437-449). Recombinant eIF5A derived from RCP (the secreted form inresponse to hypoxia/reoxygenation) had mainly the hypusinated form (FIG.2 b, right panel; spot B′), while recombinant eIF5A derived fromcytosolic fractions of untreated transfected cells had mostly theunhypusinated form (FIG. 2 b, left panel; spot A). Here, as theisoelectric points of spots A and B of cytosolic form were 5.3 and 5.4,respectively, and the isoelectric points of spots A′ and B′ of secretedform were 5.2 and 5.3, respectively, it appeared that the isoelectricpoint decreased by approx. 0.1 during conversion from an cytosolicprotein to a secreted protein. This conversion was further confirmed bytwo-color Western blotting by using the same gel in (0094)

Example 4

(4) The recombinant eIF5A derived from RCP (mainly containinghypusinated eIF5A) potently induced apoptosis of cultured cardiacmyocytes as shown by double staining with terminal deoxynucleotidyltransferase nick-end-label (TUNEL; brown color) and cardiac myosin (bluecolor) (FIG. 3 a), while the recombinant mutant eIF5A (K50A) derivedfrom RCP (unhypusinated eIF5A) only partially induced apoptosis ofcardiac myocytes (FIG. 3 a). In contrast, in the untreated control groupand the cytosolic (mostly unhypusinated) eIF5A-treated group, almost nocardiac myocytes underwent apoptosis (FIG. 3 a). FIG. 3 b shows timecourse of percentage of apoptotic cardiac myocytes induced by therecombinant eIF5A (RCP), the recombinant mutant (K50A) eIF5A (RCP), andthe recombinant (cytosolic) eIF5A. The recombinant eIF5A(RCP) alsoinduced translocation of the apoptosis-inducing factor (AIF) from thecytosol (mitochondria) to the nucleus in cultured cardiac myocytes asdetermined by double staining for AIF and Hoechst 33342 (1 μg·mL⁻¹, FIG.3 c). The recombinant eIF5A (RCP) markedly increased the cytosolicfractions of cytochrome c and the active form of caspase-3 in culturedcardiac myocytes with its peak at 48 hours (FIG. 3 d, arrow, and FIG. 3e). The induction of apoptosis of cardiac myocytes by the recombinanteIF5A (RCP) was further confirmed by Annexin-V staining (FIG. 3 f) andthe hypercondensation of nuclear chromatins by electron microscopy (FIG.3 g).

Example 3

It is known that apoptosis of mammalian cells can be classified into thecaspase-dependent pathway and the caspase-independent pathway, and thatactivation of poly (ADP-ribose) polymerase-1 (PARP-1) signals tomitochondria to release AIF mediates the apoptosis signal cascade in thecaspase-independent pathway. Accordingly, to investigate thecontribution of the caspase dependent pathway and the caspaseindependent pathway to the apoptosis of cardiac myocytes induced byrecombinant eIF5A (RCP), was analyzed the effects of PARP-1 inhibitor3-aminobenzamide (Sigma) and broad caspase inhibitor Z-VAD.fmk (BIOMOLInternational, LP) on induction of apoptosis. FIGS. 3 h and 3 i showthat PARP-1 inhibitor (3-aminobenzamide) and caspase inhibitor(Z-VAD.fmk) significantly inhibited apoptosis induction by approx. 30%and 70%, respectively, which demonstrates the contribution of the bothpathways. To confirm that the secreted eIF5A actually mediateshypoxia/reoxygenation-induced apoptosis of cardiac myocytes, the effectof removal of eIF5A from the reoxygenation-conditioned medium (RCM) byusing a neutralizing antibody on RCM-induced apoptosis of cardiacmyocytes was analyzed. FIGS. 3 j and 3 k show that removal ofRCM-derived eIF5A by using anti-eIF5A antibody (J-C) decreased apoptosisof cardiac myocytes significantly (approx. 55%), which indicates thatRCM-induced apoptosis was at least partially mediated by the secretedeIF5A. The reason why immunodepletion of RCM-derived eIF5A did notcompletely abrogate the induction of apoptosis appears that theneutralizing activity of the antibody was not strong enough tocompletely deplete eIF5A derived from RCM because the amino acidsequence of eIF5A is highly conserved among mammalians.

Example 4

(Isoelectric Point of Secreted (Hypusinated) eIF5A)

FIG. 4 shows two-dimensional electrophoresis results of the myc- andFLAG-tagged recombinant eIF5A. In the figure, A is unhypusinated eIF5A,and B is hypusinated eIF5A. Specifically, the upper panel of FIG. 4shows Western blot results of cytosolic fractions of the myc- andHis-tagged recombinant eIF5A from transfected cells not treated withRCP. From the upper panel of FIG. 4, it was found that the isoelectricpoints of the cytosolic eIF5A, that is, conventionally known eIF5A wereapprox. 5.4 for the cytosolic unhypusinated eIF5A and approx. 5.5 forthe cytosolic hypusinated eIF5A. In other words, the isoelectric pointof eIF5A is increased by hypusination by 0.1.

Furthermore, the lower panel of FIG. 4 shows Western blot results of theFLAG- and His-tagged recombinant eIF5A from reoxygenation-conditionedPBS (RCP). From the lower panel of FIG. 4, it was found that theisoelectric point of secreted eIF5A decreased by 0.1 as compared withnon-secreted (cytosolic) eIF5A. Specifically, the isoelectric point ofsecreted unhypusinated eIF5 (A′) was approx. 5.3, and the isoelectricpoint of secreted hypusinated eIF5A (B′) was approx. 5.4.

Example 5

Subcellular localization of eIF5A in cultured cardiac myocytes wasanalyzed by double immunostaining with an anti-eIF5A antibody (J-M) andanti-cardiac myosin (CMA19) antibody. Immunostaining for cardiac myosinshowed that most cells were cardiac myocytes (FIG. 5 a, lower panel).Cardiac myocytes under normoxia only weakly express eIF5A at theperinuclear region (FIG. 5 a, upper left panel). Cardiac myocytessubjected to hypoxia for 60 min and subsequent reoxygenation for 10 minclearly showed granular staining in their peripheral cytoplasm as wellas perinuclear region with the anti-eIF5A antibody (J-M) (FIG. 5 a,upper middle panel and upper right panel, arrows). Furthermore,immunoelectron microscopy with the anti-eIF5A antibody (J-M) revealedthat eIF5A existed in granules immediately adjacent to (FIG. 5 b, leftpanel) or on the plasma membrane (FIG. 5 b, middle panel), or away fromthe plasma membrane as if the granules were being secreted from thecardiac myocytes (FIG. 5 b, right panel). This strongly suggests thateIF5A can be secreted from cardiac myocytes in response tohypoxia/reoxygenation like secretary granules. No significant signal wasdetected in nonimmunized rabbit sera. To confirm the expression of eIF5Aon cardiac myocytes in response to in vivo ischemia/reperfusion, eIF5Aof ventricular tissues derived from sham-operated rats and derived fromrats subjected to myocardial ischemia/reperfusion were immunostained. Inthe sham-operated rats and the rats subjected to myocardial ischemia for30 min, eIF5A was hardly expressed on cardiac myocytes (FIG. 5 c, upperleft and right panels, respectively). Myocardial ischemia for 30 min andsubsequent reperfusion for 15 min induced weak expression of eIF5A onthe plasma membrane of some cardiac myocytes (FIG. 5 c, lower leftpanel). Myocardial ischemia for 30 min and subsequent reperfusion for 30min clearly increased the expression of eIF5A on the plasma membrane ofmany cardiac myocytes (FIG. 5 c, lower right panel). This stronglysuggests that the same mechanism is involved in cardiac response toischemia/reperfusion in vivo as to hypoxia/ reoxigeneration in vitro.

Example 6

For the intracellular molecular mechanism in cellular response to theoxidative stress, it was reported that the earliest step was activationof Src tyrosine kinases, followed by activation of Ras and Raf-1 inmammalian cells in response to ultraviolet (Cell 71, 1081-1091 [1992]).These intracellular signaling cascades were confirmed in cardiacmyocytes in response to in vitro hypoxia/reoxygenation (Circ. Res. 78,82-90 [1996]; Biochem. Biophys. Res. Commun. 226, 530-535 [1996]).PARP-1 is a nuclear enzyme known to play a role in repair of DNA damageby depleting NAD and ATP, which leads to cell death. Oxygen freeradicals cause DNA damage and thereby activate PARP-1. PARP-1 thereforemay contribute reperfusion injury of previously ischemic tissue throughgeneration of oxygen free radicals. In fact, inhibition of PARP-1activity attenuates reperfusion injury of various tissues includingmyocardium (Proc. Natl. Acad. Sci. 94, 679-683 [1997]).

Example 7

(Apoptosis-Inducing Ability of the Secreted eIF5A of the PresentInvention in Cancer Cells)

The results (TUNEL staining) of examination of the apoptosis-inducingability of the secreted eIF5A of the present invention against variouscells are shown in FIGS. 6 and 7. FIG. 6 shows that the recombinantsecreted eIF5A of the present invention (10 μg/ml, 30 to 36 hours)induced apoptosis of Hela cells, liver cancer cells, and glioblastomacells. On the other hand, apoptosis of quail muscle cells, which arenormal cells, was not induced until 72 hours.

Example 8

It was found that the recombinant (secreted, mainly hypusinated) eIF5Ainduced apoptosis of cardiac myocytes through the caspase-dependentpathway and PARP-1-dependent pathway. Production of oxygen free radicalsis known to occur within few minutes of reperfusion of the heart (Circ.Res. 61, 757-760 [1987]). Meanwhile, a marked amount of secreted eIF5Awas detected in RCP as early as approx. one minute of reoxygenation. Itis therefore considered that PARP-1 activation induced byischemia/reperfusion (or hypoxia/reoxygenation) is mediatedindependently by production of oxygen free radicals as well as bysecretion of hypusinated eIF5A.

In the case of mechanical load, it has been reported that mechanicalstretch of cardiac myocytes causes autocrine release of angiotensin II,which then induces activation of multiple intracellular signalingpathways in cardiac myocytes, resulting in cell hypertrophy (Cell. 75,977-984 [1993]). This indicates that exquisite autocrine mechanism inwhich cardiac myocytes respond and adapt to the external stress byincreasing contractile components against mechanical load throughreleasing angiotensin II in vivo. In contrast, the present inventionrevealed that cardiac myocytes respond but fail to adapt to someexternal stresses, such as strong oxidative stress, by undergoingapoptosis through releasing secreted hypusinated eIF5A. There may be abalance between apoptosis-inducting factors (for example, hypusinatedeIF5A and oxygen free radicals) and anti-apoptosis factors (for example,thioredoxin, cyclophilin A, and heat shock protein) in cellular responseto the oxidative stress.

When the oxidative stress is strong enough for cardiac myocytes tosecrete a sufficient amount of hypusinated eIF5A to cause apoptosis, thebalance can be lost. Therefore, neutralization of secreted hypusinatedeIF5A or blockade of a cell surface receptor specific for secretedhypusinated eIF5A protects cardiac myocytes from excessive (or lethal)oxidative stress, such as complete ischemia followed by reperfusion.Here, the cell surface receptor of secreted hypusinated eIF5A will beidentified.

Although eIF5A is identified as one of eukaryote translation initiationfactors (J. Biol. Chem. 251, 5551-5557 [1976]), the function of eIF5Ahas been only partially understood. As blocking of lysine/hypusinetransformation inhibited the translation initiation function and cellproliferation (Mol. Cell. Biol. 11, 3105-3114 [1991]; J. Biol. Chem.266, 7988-7994 [1991]), the translation initiation activity of eIF5A isknown to be correlated with hypusination (unique posttranslationalmodification) of the specific lysine residue of eIF5A. More recently, ithas been reported that unhypusinated eIF5A rapidly traslocates from thecytoplasm to the nucleus and mediates apoptosis in response to TNF(tumor necrosis factor)-α (Exp. Cell. Res. 313, 437-449 [2007]).Overexpression of eIF5A in cancer cells by transfection witheIF5A-expressing adenovirus caused a dramatic accumulation ofunhypusinated and deoxyhypusinated eIF5A as compared with hypusinatedeIF5A and induced marked apoptosis of the cells. Transfection with(mutant) eIF5A (K50A)-expressing adenovirus caused predominantaccumulation of unhypusinated eIF5A and similarly induced markedapoptosis of cells. It was therefore concluded that induction ofapoptosis of these cells did not arise from decreases of hypusinatedeIF5A, but rather than an accumulation of unhypusinated eIF5A, whichseems to induce apoptosis. Accordingly, it was considered thathypusinated eIF5A in cytoplasm contributes to cell proliferation,whereas translocated unhypusinated eIF5A in the nucleus mediatesinduction of apoptosis.

Therefore, the present invention demonstrated for the first time thathypusinated eIF5A is rapidly secreted from cardiac myocytes in responseto hypoxia/reoxygeneration and acts as an apoptosis-inducing ligand bybinding to some cell surface receptor on cardiac myocytes. As thesecreted unhypusinated eIF5A had significantly decreased theapoptosis-inducing activity than secreted hypusinated eIF5A did,hypusination appears to play a key role in the receptor binding andstimulation. Therefore, the present invention revealed a third mechanismin which eIF5A functions as an apoptosis-inducing ligand in an autocrinefashion in the extracellular space. As eIF5A is not essential for usualprotein synthesis, it has been thought that eIF5A may be required fortranslation of certain mRNAs or, rather be involved in other cellularmetabolisms. It is therefore considered that the extracellular functionas an apoptosis-inducing ligand, which was revealed by the presentinvention, is the primary function of this unique protein.

As eIF5A is ubiquitous and abundant among various cell types, it isevident that the autocrine mechanism plays a key role in pathogenesis ofoxidative stress-induced cell injury induced by various environmentstimuli and that involved in many common diseases caused by an oxidativestress, such as atherosclerosis, aging, and cancer.

Example 9

(Examination of Effect of Inhibition of Hypusination of eIF5A Using DHSInhibitor on Rat Myocardial Ischemia/Reperfusion Injury)

eIF5A is deoxyhypusinated by deoxyhypusine synthase (DHS) in thecytoplasm (deoxyhypusine eIF5A) and then hypusinated by deoxyhypusinehydroxylase (DOHH) (hypusinated eIF5A). eIF5A is further secretedextracellularly by an oxidative stress stimulus as secreted eIF5A andacquire the apoptosis-inducing activity. It is therefore considered thatthe apoptosis-inducing activity can be reduced by inhibitinghypusination of eIF5A with a DHS inhibitor. Accordingly, (1) theinhibitory effect on myocardial infarction was investigated byadministering GC7 (N1-guanyl-1,7-diaminoheptane), a DHS inhibitor, fromfive days to the day before myocardial ischemia/reperfusion (1 mg/kg,everyday, peritoneal administration). A group similarly to which thesolvent of GC7 was administered was established as the control group. Inan experimental system for rat myocardial ischemia/reperfusion asmentioned above, complete coronary artery occlusion was performed byclamping the left coronary artery for 30 min, then reperfusion wasperformed by removing the clamp and the rats were killed 24 hours later.The heart was horizontally sliced from the cardiac apex towards the baseof the heart and stained with 1% TTC (trimethyl tetrazolium chloride)solution to determine the extent of myocardial infarction. As shown inFIG. 8, the extent of myocardial infarction for control group wasapprox. 35.6% (representative example; FIG. 9, upper left panel),whereas that for GC7 administration group was approx. 13.4%(representative example; FIG. 8, lower left panel) and myocardialinfarction was significantly inhibited (approx. 1/2.7). Thisdemonstrated that hypusination of eIF5A plays a key role in (myocardial)ischemic reperfusion injury.

Example 10 (Examination of Inhibitory Effect on Rat MyocardialIschemia/Reperfusion Injury Using Anti-eIF5A Neutralizing MonoclonalAntibody) (Preparation of Anti-eIF5A Neutralizing Monoclonal Antibody)

Hybridomas were produced by immunizing BALB/c mice with cytosolicrecombinant eIF5A, and screened for their inhibitory effects onreoxygenation-conditioned medium (RCM)-induced apoptosis of cardiacmyocytes, to thereby obtain a single clone producing an antibody whichrecognizes cytosolic and secreted eIF5A (designated as YSC-1). Theanti-eIF5A neutralizing monoclonal antibody (YSC-1) was administered (3mg/kg, intravenous injection) at 20 min after the start of myocardialischemia (10 min before reperfusion), and its inhibitory effect on thedevelopment of myocardial infarction was investigated. The group towhich mouse IgG was similarly administered was established as thecontrol group. As shown in FIG. 9, administration of the anti-eIF5Aneutralizing monoclonal antibody (YSC-1) markedly (approx. ⅛) reducedthe extent of myocardial infarction to approx. 4.2% (representativeexample; FIG. 9, lower left panel) as compared with the control group(approx. 34.3%) (representative example; FIG. 9, upper left panel). Theabove findings suggested that secreted eIF5A conclusively mediatesischemia/reperfusion injury and that the anti-eIF5A neutralizingmonoclonal antibody therapy is very useful for prevention of theischemia/reperfusion injury.

Example 11

To investigate the action of secreted eIF5A in myocardialischemia/reperfusion injury, a direct ELISA system was developed using abiotinylated anti-eIF5A antibody (J-M) as detecting antibody andstreptavidin horseradish peroxidase (HRP), and serum secreted eIF5Alevels in rats subjected to myocardial ischemia/reperfusion wereexamined. No significant change was observed in serum secreted eIF5Alevels between the control condition (before ischemia) and at 30 minafter ischemia (before reperfusion). The serum secreted eIF5A levelssignificantly increased at 5 min after reperfusion (64.47±11.18 ng/ml),as compared with that before ischemia (4.04±2.22 ng/mL [mean±SE]:p=0.005) and that at 30 min after ischemia (4.00±1.90 ng/mL: p=0.007).The serum secreted eIF5A levels gradually decreased at 15 min afterreperfusion (FIG. 10).

1. A method for determining an oxidative stress, comprising measuring asecreted eIF5A protein in a body fluid or a tissue specimen.
 2. Adiagnostic agent for an oxidative stress, comprising a reagent formeasuring a secreted eIF5A protein.
 3. An apoptosis inhibitor,comprising a secreted eIF5A protein inhibitor.
 4. The apoptosisinhibitor according to claim 3, wherein the apoptosis is caused by anoxidative stress.
 5. A method for inducing apoptosis, comprisingadministering a secreted eIF5A protein.
 6. A method for inhibitingapoptosis, comprising administering a secreted eIF5A protein inhibitor.